Large-Scale Production, Biochemical and Structural Characterization of Lactate Dehydrogenase for the Discovery of Novel Small Molecule Inhibitors

Lactate Dehydrogenase (LDH) catalyzes the interconversion of pyruvate and lactate in the final step of anaerobic glycolysis. Many invasive tumors depend on an increased rate of glycolysis in order to maximize their growth potential under hypoxic environments, a phenomenon known as the Warburg Effect. By finding a way to inhibit LDH, contributions would be made towards the management of cancerous cells by inhibition of their energy production. Human LDH isoenzyme A was subcloned into an in-house vector (pET28a with a PreScission cleavage site) and overexpressed in E. coli Tuner(DE3) cells as a fusion protein with maltose binding protein (MBP) to increase solubility. The fusion protein was cleaved, purified, and subjected to crystallization trials using the hanging drop vapor diffusion method. Crystals of the binary complex of LDH-A with NADH were obtained, and conditions continue to be optimized in order to increase crystal size and diffraction quality. Steady-state kinetic characterization revealed Km(Pyruvate) to be 130 ± 19 μM and the specific activity to be 382 ± 16 U/mg. The IC50 value for the known weak inhibitor oxamate was determined to be 65 ± 2 μM. Future plans include high-throughput screening (HTS) for novel LDH-A inhibitors, co-crystallization of LDH-A with HTS hit compounds, and the structure-based design of potent and specific inhibitors of this enzyme